3-spiro-cyanin fluorochromes and their use in bioassays

ABSTRACT

The invention concerns sterically shielded, stabilized fluorescent dyes based on symmetrical or unsymmetrical 3-spirocyanines and their utilization as marker-molecules in analytical procedures. The fluorescent dyes according to the invention are characterized by a strongly reduced proneness towards aggregation due to the insertion of bulky spiro-substituents and thus enable to reach a high degree of coupling to the target molecule without a significant loss of fluorescence due to self quenching effects. They therefor exhibit a high quantum yield and are thus excellently suitable as fluorescent dyes, especially as NIR-fluorescent dyes, for the coupling or binding to biological and non-biological target molecules used in analytical methods. The analytical methods comprise all methods in which fluorescence-optical methods are used to detect biological and non-biological molecules.

The invention concerns sterically shielded, stabilized fluorescent dyeson the basis of symmetrical or unsymmetrical 3-spirocyanines and theirapplication as marker-molecules in analytical methods.

Fluorescent dyes are increasingly used to label enzymes, antibodies andnucleic acids and employed in immunoassays, fluorescence microscopy andfor sequencing. Traditionally, fluorescent dyes are used which areexcited using light from the UV or visible region of the electromagneticspectrum. Their excitation and emission spectra overlap with the givennatural serum fluorescence, making them only partially suitable foranalysis of natural samples (e.g. blood and serum samples, cells).Fluorescent dyes with excitation wavelength in the near infrared regionof the light spectrum are thus used since a couple of years, allowingthe measurement of biological samples with low background fluorescenceand concomitantly increased sensitivity.

The demand of biochips (protein-, DNA-arrays) that enable the specificdetection of proteins as well as DNA or fragments thereof, growsexponentially due to scientific developments in the proteomics andgenomics sector. Most of these applications also involve the use offluorescent dyes.

The use of reactive and water soluble NIR-fluorophores for coupling withbiomolecules was first described in U.S. Pat. No. 5,268,486. Chemicalgroups are inserted, preferably as N-substituents, that are suitable forcoupling with reactive amino acid side chains (e.g. amino-, thio-,carbonyl-, or hydroxy groups), some of these chemical groups beingisothiocyanates, thiocyanates, hydrazines, hydroxy-succinimidylesters,disulfides etc. This principle was extended in recent time beyondsymmetrical cyanomethine dyes (2,2′-indo-cyanines) to encompassstructurally related merocyanines and styrylcyanine dyes.

All dyes of the cyanine type as well as other commonly used fluorescentdyes, e.g., rhodamines, show a characteristic behavior in aqueoussolution. Due to the planar chemical structure (arrangement) of thefluorophores, aggregation or dimer-formation occurs especially at highercoupling ratios (molar ratio dye/protein; D/P>5). This leads todrastically reduced fluorescence due to radiation free intermoleculartransition processes. This phenomenon arises in solutions, e.g. inpresence of high concentrations of salts, as well as with protein boundfluorophores. Dyes without additional hydrophilic groups andamino-substituted cyanine dyes are especially sensitive to this problem.

The dyes described in U.S. Pat. No. 5,268,486 and DE 39 120 46 couldonly reduce but not eliminate this problem through insertion ofarylsulfonates to raise their water solubility. In general, the quantumyield in aqueous solutions, however, is reduced significantly comparedto organic solvents.

Many different interactions are utilized for the labelling ofoligonucleotides or DNA/RNA. Besides the classical intercalation dyeslike ethidium bromide, the specific ionic binding to phosphate groups ofnucleotides (U.S. Pat. No. 5,410,030) by positively charged dyes andvarious covalent couplings of dyes to modified purin/pyrimidinbases(U.S. Pat. No. 6,027,709) as well as to phosphate groups of nucleotidesare feasible. This development reflects the high increase in demand offluorescent dyes for DNA labelling and sequencing.

It is the task of the presented invention to suggest fluorescent dyesthat exhibit a high quantum yield and simultaneously a low tendencytowards aggregation. Furthermore, these fluorophores shall cover a widevariety of different ways to bind to a vast number of target moleculesand thus allow for a wide variety of application fields.

The task is solved by the characteristic features described in claim 1.The other dependant claims show further advantageous refinements(embodiments).

The application of fluorophores according to the invention is describedby claims 1 to 13. Fluorescent dyes are suggested according to thisinvention, which are based on a basic structure of symmetrical orunsymmetrical cyanine dyes with at least one sterically demandingspiro-substituent in 3-position of the indole headgroup. Thesefluorescent dyes will be referred to as spironines. The fluorescent dyesaccording to the invention have the following basic structure:

Fluorescent dye of the general formula A-Z-A′ with

A a group of general formula I

I

wherein X is

O, S, SO, SO₂ or

W⁻=any counter ion, preferably halogenide, perchlorate or tosylate.

A′ is selected from a group having the general formulas II to V,

wherein Y is O, S, NR₁₇,

and T is

O, S, SO, SO₂ or

Z represents a group with the general formula VI or VII

with n′ is an integer selected from the group consisting of 0, 1, 2, or3 orwherein at least one of the residues R₁ to R₂₆ is selected from a groupconsisting ofa) a chemical reactive group for covalent coupling to a target moleculeorb) an ionic group for coupling to a target molecule by ionic interactionforces orc) a lipophilic group for coupling to a target molecule by adsorption.

By means of the selective introduction of the groups a) to c) into thefluorescent dye, it is made possible to bind the dye to different targetmolecules and thus allow for different fields of applications.

The other groups are chosen independently from each other from thegroups H, alkyl (C₁-C₁₀), alkoxy (C₁-C₁₀), trifluoromethyl, halogen,sulfonic acid, sulfonate, phosphoric acid and phosphonate.

Through use of cyanine dyes with bulky substituents in 3-position of theindole headgroup, especially cyclic substituents, so called spirones,e.g. spiro-1′-cyclohexan, spiro-4′-1′-piperidine,4′-1′-tetrahydropyrane, spiro-4′-1′-tetrahydrothipyrane,spiro-4′-1′-oxotetrahydro-thiopyrane,spiro-4′-1′,1′-dioxotetrahydrothiopyrane, the central polymethine chainis shielded extensively.

Surprisingly, it could be shown according to the invention thatfluorescent dyes substituted like this exhibit a significant reducedtendency towards aggregation. Furthermore, a higher photostabilitycompared to commercially available cyanine dyes was observed.Particularly interesting in this matter is the utilization of cyclicsubstituents, e.g. substituted spiro-cyclohexanes, spiro-piperidines,spiro-tetrahydrooxopyranes or spiro-tetrahydrothiopyranes. Based onthese spiro compounds, a new elegant route is created to control thewater solubility by choosing a polar or non-polar substituent as well asto positively influence the binding behavior of the dye by insertion ofreactive groups R_(x) or ionic groups. The shielding and thusstabilizing effect of the spiro groups can be amplified through furtherinsertion of bulky substituents at the spiro-residues.

The invention based fluorophores can be excited by light of a wavelengthof 600-1000 nm. Typical for the fluorescent dyes are high molarextinction coefficients and very high quantum yields. The stokes shiftnormally is at least 15 nm. A utilization of these dyes in a broad fieldof applications is made possible by cost effective laser diodes emittinglight in the range of 670-830 nm.

The groups R₁, R₄, R₆ to R₁₀, R₁₃ and R₁₇ to R₂₁ in the general formulasI to V are of higher significance. These positions are especiallysuitable for insertion of substituents to increase the water solubility,the insertion of non-polar substituents to increase the lipophily and/orat least one reactive group R_(x) for the coupling to biomolecules.

Thus, the fluorescent dyes contain at least one reactive group R_(x) forthe coupling to target molecules in the general formulas I to VIIadvantageously as groups R₁, R₄, R₆ to R₁₀, R₁₃ and R₁₇ to R₂₁,particular advantageously s groups R₁, R₆ to R₁₀ and R₁₈ to R₂₁ andparticular advantageously as groups R₆ to R₉ and R₁₈ to R₂₁.

The reactive group R_(x) can be covalently bound directly to the dye orthrough a bridge made by several atoms and exhibits a suitablechemistry.

The reactive group R_(x) is preferentially chosen from the group ofcarboxylic acids, activated esters, acylazides, acylhalogenides,acylnitriles, aldehydes, anhydrides, arylamides, alkyl halides,anilines, alkylsulfonates, arylhalogenides, thioles, azides, aziridines,borates, carbodiimides, diazoalkanes, epoxides, glyceroles,haloacetamines, halotriazines, hydrazines, hydroxylamines, imidoesters,isocyanates, isothiocyanates, maleimides, phosphoramidites, silylhalides, sulfonates and sulfonylchlorides. Subsequently some exemplarygroups R_(x) and their structures are shown.

activated carboxylic acid

with n, m and p independently from each other equal to 1 to 8carbodiimides

with n=1 to 8anhydrides

with n=1 to 8carboxylic acid azides

With n=1 to 8

Coupling of nucleophiles through

epoxides

with n=1 to 8isothiocyanates—(CH₂)_(n)—NCS

with n=1 to 8isocyanates—(CH₂)_(n)—NCO

with n=1 to 8aziridines

with n=1 to 8

maleimides

with n=1 to 8pyridyl-disulfide activated groups

with n and m independently from each other equal to 1 to 8halodi- and triazines

with W=chlorine or bromine and n=1 to 8vinylsulfones

with n=1 to 8acylimidazoles

with n=1 to 8phosphoramidite

with n=1 to 8

Tab. 1 shows an overview over possible ways of creating dye-conjugatesby covalent binding.

Additionally, the fluorescent dye according to the invention may containat least one ionic group. Some of the preferred ionic groups for exampleare selected from the group of carboxylic acids, sulfonic acids,sulfonates, phosphoric acids, phosphonates, phosphordiesters,phosphortriesters and primary to quaternary amines. Advantageously atleast one of the groups R₁, R₄, R₆ to R₁₀, R₁₃ and R₁₇ to R₂₁,particular advantageously at least one of the groups R₁, R₆ to R₁₀ andR₁₈ to R₂₁ and advantageously at least one of the groups R₆ to R₉ andR₁₈ to R₂₁.

Further, one group of the group A, chosen from the group R₁, R₆ to R₉,and one group of the group A′, chosen from the group R₁₀, R₁₇ to R₂₁,can represent ionic groups with opposite charges. This has the specialadvantage that an additional stabilization of the fluorescent dye ismade possible by this intramolecular ionic bridge. It is also possiblefor the ionic groups in the groups A and A′ to have the same charge andto form a complex with an ion of opposite charge. It is preferred herebyto have two anionic groups that form a complex with a metal-ion fromthe 1. to 3. main- or subgroup.

To raise the solubility in water, for example groups like carbon acids,carbohydrates, sulfonic acids, sulfonates, phosphates, phosphonates,amines, halogens, polyoles or polyethers could be chosen and inserted atany position of the fluorescent dye, advantageously as at least one ofthe groups R₁, R₄, R₆ to R₁₀, R₁₃ and R₁₇ to R₂₁, particularadvantageously as at least one of the groups R₁, R₆ to R₁₀ and R₁₈ toR₂₁ and most particular advantageously as at least one of the groups R₆to R₉ and R₁₈ to R₂₁.

To raise the lipophily, long-chain saturated or unsaturated alkyl groups(preferably C₆ to C₁₈) or fatty acids/fatty alcohols could be chosen andinserted at any position of the fluorophor, advantageously at least oneof the groups R₁, R₄, R₆ to R₁₀, R₁₃ and R₁₇ to R₂₁, particularlyadvantageous at least one of the groups R₁, R₆ to R₁₀ and R₁₈ to R₂₁ andespecially advantageous at least one of the groups R₆ to R₉ and R₁₈ toR₂₁.

In another advantageous embodiment of the invention two ortho residuesat the aromatic ring may be combined to form at least one additionalaromatic, carbo- or heterocyclic ring.

The dye can form complexes or conjugates with a biological ornon-biological target molecule by creation of one or more covalent bondsusing one or more groups R_(x), the target molecule preferably beingfrom the group of antibodies, proteins, peptides, enzyme substrates,hormones, lymphokines, lipids, phospholipids, metabolites, receptors,antigenes, haptenes, lectines, toxines, carbon hydrates,oligosaccharides, polysaccharides, nucleic acids, desoxyribonucleicacids, derivatizid desoxyribonucleic acid, derivatized nucleic acids,DNA-fragments, RNA-fragments, drugs, virus particles, virus components,yeast, yeast components, bacteria, bacteria components, blood cells,blood cell components, biologic cells, non-cellular blood components,poisons, polymers, polymer particles, glass particles, glass surfaces,plastic surfaces, plastic particles, polymer membranes, metals,conductors or semiconductors.

In another advantageous refinement fluorescent dyes are bound to thesurface of or incorporated into nano- or microparticles, mostly on thebasis of polymer materials. These particles can then be usedadvantageously in analytical methods.

The fluorescent dyes according to the invention especially distinguishthemselves from the state of the art by a strongly reduced pronenesstowards aggregation due to the insertion of bulky spiro-substituents andthus a high degree of coupling to the target molecule withoutself-quenching of the fluorescence. They therefor exhibit a high quantumyield and are thus excellently suitable as fluorescent dyes, especiallyas NIR-fluorescent dyes, for the coupling or binding to biomoleculesused in bioassays. The analytical methods comprise all methods in whichfluorescence-optical methods are used to detect biomolecules. Apreferred way of realization are fluorescence-immuno-tests which arebased on known biochemical assays of general receptor-ligand systems,for example antibody-antigen, lectin-carbohydrate, DNA orRNA-complimentary nucleic acids, DNA or RNA-proteins, hormone receptors,enzyme-enzym co-factors, protein G or protein A-immunglobulin oravidin-biotin.

If the fluorescent dye according to the invention contains adequatenucleophilic groups, preferably amino-, thio-, or hydroxy-groups, acoupling of the dye, for example after activation as phosphoramidite, toa nucleotide becomes possible. This process is especially important forthe preparation of dye-labelled (desoxy-)nucleotides, which for examplecan be used in sequencing machines.

Some representative fluorescent dyes according to the invention aresummarized in the following.

with M⁺=metal cation and with X, T, and n′ as previously defined.

with M+=metal cation, NHS=N-Hydroxy-succinimide and with X, T, W⁻ and n′as previously defined.

with X, T, W⁻ and n′ as previously defined, m is equal to 0, 1-17.

with M⁺=metal cation, X, Y, T, W⁻, m and n′ as previously defined.

with X, Y, T, W⁻, m and n′ as previously defined, p is equal to 1 to 8.

with X and n′ as previously defined, q is equal to 1 to 8.

with M⁺=metal cation and n′ as previously defined,NHS=N-hydroxy-succinimid, r and q independently from each other equal to1 to 8.

with W⁻ and n′ as previously defined.

with W⁻ and n′ as previously defined.

with W⁻ and n and n′ as previously defined, NHS=N-hydroxy-succinimide,PEG=poly ethylen glycole and with t and u independently from each otherequal to 1 to 8.

with W⁻ and n′ as previously defined, R′=trityl, R″=phosphoramidite, vand w independently equal to 0 to 6.

with M⁺=metal cation, X, Y, T, W⁻ and n′ as previously defined.

with M⁺=metal cation, X, Y, T, W⁻ and n′ as previously defined, z equalto 0, 1 to 6.

The invention will be described in more detail by way of figures andexamples below, without restricting it to them.

FIG. 1 Synthesis of compounds S1 to S3.

(Part 1)

FIG. 2 Synthesis of compounds S1 to S3.

(Part 2)

FIG. 3 Synthesis of compounds S4 to S7.

(Part 1)

FIG. 4 Synthesis of compounds S4 to S7.

(Part 2)

FIG. 5 Synthesis of compound S8.

FIG. 6 Synthesis of compound S9.

(Part 1)

FIG. 7 Synthesis of compound S9.

(Part 2)

FIG. 8 Absorption spectrum of the fluorescent dyes (compounds S1 to S3)according to the invention.

FIG. 9 Absorption- and emission-spectrum of compound S2.

FIG. 10 Absorption- and emission spectrum of the protein conjugateaccording to example 10.

FIG. 11 Photostability of compound S3 (according to example 3).

FIG. 12 Fluorescence of the protein conjugate (compound) according toexample 10 depending of the incubation time.

FIG. 13 Fluorescence of compound in the Fluorescence of compound inpresence of high protein concentrations presence of high proteinconcentrations.

PREPARATION OF DYES OF THE SYMMETRICAL 3-SPIRO-1′-CYCLOHEXAN INDOLE TYPEExample 1 Synthesis of Dye S1 Preparation of2-methyl-3-spiro-1′-cyclohexan-5-sulfo-3H-indole; Potassium Salt (FIG.1, 1 a)

30 mmol (5.646 g) phenylhydrazine sulfonic acid is heated with 60 mmol(7.572 g) cyclohexylmethylketone and 60 mmol (5.889 g) potassium acetatein 30 ml glacial acetic acid for 6 h under reflux. Subsequently thevolatile components were evaporated under reduced pressure. The brownresidue is transferred into an extraction thimble and extracted with 200ml 2-propanole using a Soxleth apparatus. The product startscrystallising in the flask even in the heat, after cooling it isfiltered with suction and dried in vacuum.

Yield: 6.044 g (66%)

Preparation of1-(5-carboxypentyl)-2-methyl-3-spiro-1′-cyclohexan-5-sulfo-3H-indoliumbromide; potassium salt (1 b)

4.5 mmol (1.418 g) 2-methyl-3-spiro-1′-cyclohexan-5-sulfo-3H-indole ismixed with 5.5 mmol (1.073 g) 6-bromohexanoic acid and reacted in themelt at 110° C. for 2.5 h. After cooling the mixture is triturated withacetone, filtered, repeatedly washed with acetone and finally dried invacuum.

Yield: 1.505 g (66%)

The quaternary spiro-indole (2 c) is the key component for the furthersteps in the synthesis.

Dye S11-(5-carboxypentyl)-2-[5-(1-(5-carboxypentyl)-1,3-dihydro-3-spiro-1′-cyclohexan-5-sulfo-2H-indole-2-yliden)-propenyl]-3-spiro-1′-cyclohexan-5-sulfo-3H-indoliumhydroxide, inner salt, potassium salt: (FIG. 2, 1 c)

0.4 mmol (205 mg)1-(5-carboxypentyl)-2-methyl-3-spiro-1′-cyclohexan-5-sulfo-3H-indoliumbromide (potassium salt) together with 1 mmol (148 mg) triethoxymethanein 2 ml pyridine are heated under reflux for 30 minutes. After coolingthe crude dye is precipitated with ether and purified by preparativeHPLC using RP-18 and ethanol:water gradient.

According to this procedure cyanine dyes with longer polymethine chainare also accessible.

Example 2 Synthesis of Dye S21-(5-carboxypentyl)-2-[5-(1-(5-carboxypentyl)-1,3-dihydro-3-spiro-1′-cyclohexan-5-sulfo-2H-indole-2-yliden)-penta-1,3-dienyl]-3-spiro-1′-cyclohexan-5-sulfo-3H-indoliumhydroxide, inner salt, potassium salt: (FIG. 2, 1 d)

0.22 mmol (111.6 mg)1-(5-carboxypentyl)-2-methyl-3-spiro-1′-cyclohexan-5-sulfoindoliumbromide (potassium salt) were dissolved in 1 ml of dry pyridine andrefluxed. Then 0.5 ml tetraethoxypropane is added in portions over aperiod of 2.5 h. The precipitated dye is repeatedly washed withpyridine, finally washed with diethylether and purified by preparativeHPLC using methanol water gradient on silicagel RP-18.

Example 3 Synthesis of Dye S31-(5-carboxypentyl)-2-[5-(1-(5-carboxypentyl)-1,3-dihydro-3-spiro-1′-cyclohexan-5-sulfo-2H-indole-2-yliden)-hepta-1,3,5-trienyl]-3-spiro-1′-cyclohexan-5-sulfo-3H-indoliumhydroxide, inner salt, potassium salt (FIG. 2, 1 e)

0.32 mmol (125 mg)1-(5-carboxypentyl)-2-methyl-3-spiro-1′-cyclohexan-5-sulfoindoliumbromide (potassium salt) together with 0.16 mmol (45 mg) glutaconedianilhydrochloride and 0.65 mmol (63 mg) potassium acetate in a mixture of 2ml acetic anhydride and 0.5 ml glacial acetic acid are heated to refluxfor 30 minutes. The crude dye is precipitated with acetone, filtered offand purified by preparative HPLC using a ethanol-water gradient onsilicagel RP-18.

Preparation of Dyes of the Asymmetric 3-spiro-1′-cyclohexan indole typeExample 4 Synthesis of Dye S4 2-methyl-3-spiro-1′-cyclohexan-3H-indole(FIG. 3, 2 a)

1.25 mol (135.2 g) phenylhydrazine and 1.5 mol (189.3 g)cyclohexylmethylketone in 1.25 l glacial acetic acid are heated toreflux for 3 h. The glacial acetic acid is evaporated in vacuum, 1 l ofwater is added to the residue and the solution is extracted 4× withether. After the etheric layer is washed with sodium hyrogencarbonatesolution and dried with sodium sulfate, the ether is evaporated and theresidue is distilled in vacuum.

bp. (2 mbar): 133-136° C.

yield: 192.6 g (77%)

1-(4-sulfobutyl)-2-methyl-3-spiro-1′cyclohexan-3H-indolium hydroxideInner Salt (FIG. 3, 2 b)

28.7 mmol (5.72 g) 2-methyl-3-spiro-1′-cyclohexan-3H-indole and 32 mmol(4.36 g) butanesultone are heated to 120° C. for 4 h. After cooling themixture is triturated with 50 ml ether, washed 2× with ethylacetate,again with ether and finally dried in vacuum.

Yield: 7.93 g (82%)

2-(4-acetanilino-1,3-butadienyl)-3-spiro-1′-cyclohexan-1-(4-sulfobutyl)-3H-indoliumhydroxide inner salt (FIG. 3, 2 c)

4mmol (1.34 g)1-(4-sulfobutyl)-2-methyl-3-spiro-1′cyclohexan-3H-indolium hydroxideinner salt and 5 mmol malondianil (prepared from 5 mmol (1.62 g)malondianilhydroperchlorate and 5 mmol (490 mg) potassiumacetate in abs.ethanol, diluted with diethylether and evaporation of the filtrate) in 5ml acetic anhydride and 5 ml glacial acetic acid are heated at 80° C.for 3 h. The solvent is evaporated in vacuum and the residue trituratedwith ether, filtered off and washed with ether until the filtrate iscolourless. The product is dried in vacuum and used for the next stepswithout further purification.

Yield: 2.00 g (98% d.Th.)

1-(4-acetoxybutyl)-2-methyl-3-spiro-1′-cyclohexan-3H-indolium iodide(FIG. 3, 2 d)

0.27 mol (49.8 g) 2-methyl-3-spiro-1′-cyclohexan-3H-indole and 0.27 mol(70.0 g) 4-iodobutylacetate are heated for 5 h at 110° C. After coolingthe residue is dissolved in small amount of methylenehloride and stirredwith 500 ml of ether. After stirring for a while the productcrystallises, it is sucked of, washed with ether and dried in vacuum.

Yield: 104.9 g (87%)

Analogous to the preparation of1-(4-acetoxybutyl)-2-methyl-3-spiro-1′-cyclohexan-3H-indolium iodide (2d) the according derivatives of 2-methylbenzoxazole,2-methylbenzothiazole, 4-methylpyridine and 4 methylchinoline areaccessible.

1-(4-acetoxybutyl)-2-methyl benzoxazolium iodide (2 e)

10 mmol (1.33 g) 2-methylbenzoxazole and 10 mmol (2.42 g)4-iodobutylacetate are heated for 6 h at 130° C. and after cooling mixedwith 10 ml of ethylacetate. The crystals obtained are washed with etherand dried in vacuum.

Yield: 2.17 g (58%)

1-(4-acetoxybutyl)-2-methyl benzothiazolium iodide (2 f)

10 mmol (1.49 g) 2-methyl-benzothiazole and 10 mmol (2.42 g)4-iodobutylacetate are heated for 4 h at 120° C. and after cooling mixedwith ether sucked off, again washed with ether and dried in vacuum.

Yield: 3.01 g (77%)

1-(4-acetoxybutyl)-4-methyl-pyridinium iodide (2 g)

10 mmol (0.93 g) γ-picoline and 10 mmol (2.42 g) 4-iodobutylacetate areheated 4 h at 120° C. and after cooling repeatedly stirred with ether,decanted a finally dried in vacuum.

Yield: 2.35 g (70% d.Th.) viscous mass

1-(4-acetoxybutyl)-4-methyl-chinolinium iodide (2 h)

10 mmol (1.43 g) lepidine and 10 mmol (2.42 g) 4-iodobutylacetate areheated 4 h at 120° C. and after cooling repeatedly stirred with ether,decanted a finally dried in vacuum.

Yield: 3.13 g (81% d.Th.) viscous mass

Dye S41-(4-hydroxybutyl)-2-[5-(1,3-dihydro-1-(4-sulfo-butyl)-3-spiro-1′-cyclohexan-2H-indole-2-ylidene)-penta-1,3-dienyl]-benzoxazoliumhydroxide inner salt (FIG. 4, 2 i)

0.20 mmol (101 mg)2-(4-acetanilino-1,3-butadienyl)-3-spiro-1′cyclohexan-1-(4-sulfobutyl)-3H-indoliumhydroxide inner salt, 0.21 mmol (79 mg) 1-(4-acetoxybutyl)-benzoxazoliumiodide and 0.21 mmol (29.3 μl) triethylamine are refluxed in 1 ml abs.ethanol for 30 min. After cooling 5 ml ether is added and decanted. Theresidue is dissolved in a mixture of 1 ml conc. HCl and 9 ml methanoland left overnight at 4° C. The solvent is evaporated in vacuum and theresidue is purified by chromatography on silicagel RP-18 andacetonitrile-water (1:1).

Example 5 Synthesis of Dye S51-(4-hydroxybutyl)-2-[5-(1,3-dihydro-1-(4-sulfo-butyl)-3-spiro-1′-cyclohexan-2H-indole-2-yliden)-penta-1,3-dienyl]-benzothiazoliumhydroxide inner salt (FIG. 4, 2 j)

Analogously to the procedure cited above 0.21 mmol (82 mg)1-(4-acetoxybutyl)-benzothiazolium iodide are reacted with theappropriate reagents.

Example 6 Synthesis of Dye S61-(4-hydroxybutyl)-4-[5-(1,3-dihydro-1-(4-sulfobutyl)-3-spiro-1′-cyclohexan-2H-indole-2-yliden)-penta-1,3-dienyl]-pyridiniumhydroxide inner salt (FIG. 4, 2 k)

Analogously to the procedure cited above 0.21 mmol (71 mg)1-(4-acetoxybutyl)-pyridinium iodide are reacted with the appropriatereagents.

Example 7 Synthesis of Dye S71-(4-hydroxybutyl)-4-[5-(1,3-dihydro-1-(4-sulfobutyl)-3-spiro-1′-cyclohexan-2H-indole-2-yliden)-penta-1,3-dienyl]-chinoliniumhydroxide inner salt (FIG. 4, 2 l)

Analogously to the procedure cited above 0.21 mmol (81 mg)1-(4-acetoxybutyl)-chinolinium iodide are reacted with the appropriatereagents.

Due to the state of the art, the hydroxyl substituted fluorophores,prepared according to 2 i-2 l may be converted to the correspondingphosphoramidites with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramiditeand coupled with DNA.

Preparation of Dyes of the Asymmetric3-spiro-1′-4-tetrahydropyrane-indole Type Example 8 Synthesis of Dye S82-methyl-5-sulfo-3-spiro-4′-tetrahydropyrane-3H-indole (FIG. 5, 3 a)

40mmol (7.53 g) phenylhydrazine-4-sulfonic acid and 44 mmol4-acetyltetrahydropyrane in 40 ml glacial acetic acid were refluxed 48 hunder nitrogen. Afterwards the precipitate is filtered and the aceticacid is evaporated. The residue is dissolved in a small amount of waterand 4 ml of 10M NaOH is added. After evaporation to dryness the residueis extracted with 2-propanole. The extract is evaporated and the residuedried in vacuum.

Yield: 6.94 g (57%)

1-ethyl-2-methyl-5-sulfo-3-spiro-4′-tetrahydropyrane-3H-indoliumhydroxide inner salt (FIG. 5, 3 b)

5.2 mmol (1.58 g)2-methyl-5-sulfo-3-spiro-4′-tetra-hydropyrane-3H-indole and 5.72 mmol(882 mg) diethylsulfate are heated for 5 h at 110° C. in 3 ml1.2-dichlorobenzene. After cooling the mixture is diluted with acetoneand filtered. The resulting cake is thoroughly washed with acetone anddried in vacuum.

Yield: 1.08 g (67%)

1-(5-carboxypentyl)-2-methyl-5-sulfo-3-spiro-4′-tetrahydropyrane-3H-indoliuminner salt (FIG. 5, 3 c)

10 mmol (3.03 g) 2-ethyl-5-sulfo-3-spiro-4′-tetrahydro-yrane-3H-indoleand 15 mmol (22.93 g) 6-bromohexanoic acid are heated in 10 ml of1,2-dichlorobenzene for 24 h at 120° C. After cooling the solution isdiluted with acetone and decanted. The precipitate is triturated withhot 2-propanole, cooled, filtered, washed with 2-propanole and acetoneand finally dried in vacuum.

Yield 3.07 g (78%)

2-(4-acetanilino-1,3-butadienyl)-1-ethyl-3-spiro-4′-tetrahydropyrane-5-sulfo-3H-indoliumhydroxide inner salt (FIG. 5, 3 d)

3mmol (0.93 g)1-ethyl-2-methyl-3-spiro-4′-tetrahydro-pyrane-5-sulfo-3H-indoliumtoluenesulfonate and 3.6 mmol malondianile (prepared from 3.6 mmol(prepared from 3.6 mmol (1.165 g) malondianilehydroperchlorate and 3.6mmol (353 mg) potassium acetate in abs. ethanol, diluted withdiethylether and evaporation of the filtrate) in 15 ml of a mixture ofglacial acetic acid and acetic anhydride (1:1 v/v) and heated for 3 h at80° C. The solvent is evaporated in vacuum and the residue trituratedwith ether, filtered off and washed with ether until the filtrate iscolourless.

The product is dried in vacuum.

Yield: 1.33 g (92%)

Dye S81-(5-carboxypentyl)-2-[5-(1,3-dihydro-1-ethyl)-3-spiro-4′-tetrahydropyrane-5-sulfo-2H-indole-2-yliden)-penta-1,3-dienyl]-3-spiro-4′-tetrahydropyrane-5-sulfo-3H-indoliumhydroxide inner salt potassium salt, (FIG. 5, 3 e)

2.5 mmol (1.20 g)2-(4-acetanilino-1,3-butadienyl)-3-spiro-4′-tetrahydropyrane-1-ethyl-5-sulfo-3H-indoliumhydroxide inner salt in a mixture of 5 ml pyridine and 5 ml aceticanhydride are treated with 2.7 mmol (1.07 g)1-(5-carboxypentyl)-2-methyl-5-sulfo-3-spiro-4′-tetrahydropyran-3H-indoliuminner salt and refluxed for 40 min. The solution is concentrated invacuum to one third and the crude dye is precipitated with ether anddecanted. The residue is dissolved in a small amount of 1M HCl andrepreciptated with a saturated solution of KCl. The precipitate isseparated, dissolved in water and purified by reversed-phasechromatography on silicagel RP-18 with an acetonitrile-water gradient.

Preparation of dyes of the asymmetric 3-spiro-1′-4′-piperidyl-indoletype Example 9 Syntheses of Dye S9 Synthesis of2-methyl-3-spiro-4″-(1′-carboxybenzyl)-piperidine-3H-indole (FIG. 6, 4a)

4.31 g (16.5 mmol) of the piperidylketone and 1.62 g (1.48 ml, 15 mmol)phenylhydrazine are dissolved in 15 ml glacial acetic acid under inertgas atmosphere and refluxed for 3.5 h. The excess acetic acid isdistilled in vacuum on a rotary evaporator remaining a brown oil. Theoil is taken up in 80 ml of water and extracted thrice with each 40 mldiethylether. The combined organic layers are washed once again with 40ml of water and dried over magnesiumsulfate. The solvent is distilledoff in vacuum on a rotary evaporator. The remaining brown oil ispurified by column chromatography (dichloromethane/ethylacetate=2:1) Theindole is obtained as reddish oil.

Yield=3.23 g, 9.7 mmol, (64%).

Synthesis of1-ethyl-2-methyl-3-spiro-4′-(1′-carboxybenzyl)-piperidine-3H-indoleniumethyl sulfate (FIG. 6, 4 b)

Under inert gas atmosphere 2.06 g (6 mmol) of2-methyl-3-spiro-4′-(1′-carboxybenzyl)-piperidine-3H-indole (4 a) aredissolved in 3 ml toluene. 1.02 g (0.89 ml, 6.6 mmol) diethylsulfate areadded through a syringe. The mixture is refluxed for 3.5 h,precipitating a dark purple oil, which turns solid on cooling. Thesupernatant liquid is decanted and the residue repeatedly washed withsmall portions of toluene. The solid is purified by columnchromatographywith the aid of a gradient system (1. ethylacetate, 2. acetone/glacialacetic acid=10:2). The quaternary indole is eluted twice with thesolvent and the product is obtained as highly viscous oil (0.93 g, 1.9mmol, 32%).

Synthesis of2-(4-acetanilino-1,3-butadienyl)-1-ethyl-3-spiro-4′-(1′-carboxybenzyl)-piperidine-3H-indoleniumethyl sulfate (FIG. 6, 4 c)

0.93 g (1.9 mmol) of the quaternary indole (4 b) and 2.4 mmolmalondianil (prepared from 0.79 g (2.4 mmol)malondianil-hydroperchlorate and 0.23 g (2.4 mmol) potassium acetate inabs. ethanol, diluted with diethylether and evaporation of the filtrate)in 3 ml of acetic anhydride and 3 ml glacial acetic acid are heated for3 h at 80° C. The solvent is removed under reduced pressure on a rotaryevaporator. The solid residue is triturated with diethylether, filteredand washed with ether until the filtrate is colourless. The crudeproduct (1.19 g, 1.8 mmol, 97%) is dried in vacuum and used in the nextstep without further purification.

Synthesis of1-carboxypentyl-2-methyl-3-spiro-4′-(1′-carboxybenzyl)-piperidine-3H-indoliumbromide (FIG. 6, 4 d)

2.06 g (6 mmol)2-methyl-3-spiro-4′-(1′-carboxy-benzyl)-piperidine-3H-indole (4 a) areheated in 5 ml 6-bromohexanoicacid 3.5 h a 120° C. The supernatant isdecanted from the reddish solid. Purification is carried out by columnchromatography (water/acetic acid=10:2). The quaternary indole isobtained as reddish highly viscous oil (0.95 g, 1.8 mmol, 30%).

Dye S 9 Synthesis of1-(5-carboxypentyl)-2-[5-(1,3-dihydro-1-(1-ethyl)-3-spiro-4′-(1′-carboxybenzyl)-piperidine-2H-indole-2-yliden)-penta-1,3-dienyl]-3-spiro-4′-(1′-carboxybenzyl)-piperidin-3H-indoleniumethyl sulfate (FIG. 6, 4 e)

0.62 g (1.8 mmol) of the hemicyanine (4 c) and 0.71 g (1.8 mmol) theindole (4 d) in 3 ml acetic anhydride and 3 ml pyridine are refluxed for30 min. The mixture is evaporated to dryness in vacuum on a rotaryevaporator. The solid residue is taken up in a mixture of 10 ml conc.HCl and 100 ml methanol and left overnight at 4° C. The solvent isdistilled off at reduced pressure and the residue is purified by columnchromatography (isopropanol:water, 2:1 (v/v)). The dye S9 is obtained asdark blue crystalline solid (0.36 g, 0.38 mmol, 21%).

Synthesis of1-(5-carboxypentyl)-2-[5-(1,3-dihydro-1-(1-ethyl)-3-spiro-4′-piperidine-2H-indole-2-ylidene)-penta-1,3-dienyl]-3-spiro-4′-piperidine-3H-indoleniumethylsulfate (FIG. 7, 4 f)

0.36 g (0.38 mmol) of the dye (4 e) is dissolved under an inertgasatmosphere in 5 ml of chloroform. With a syringe 0.09 g (0.07 ml, 0.46mmol) of iodotrimethylsilane is added and the mixture is stirred at roomtemperature until the educt is not detectable again by thinlayerchromatography (isopropanol/water=10:2, v/v). After that 2 ml ofmethanol is added and the mixture is stirred for 5 min. at roomtemperature. The liquid components are distilled off in vacuum on arotary evaporator and the solid residue is purified by columnchromatography with the eluent isopropanol:water=2:1. Yield: 0.23 g, 0.3mmol (80%).

The deprotected dye S9 (4 f) can be converted into a polyethylenglycole-substituted dye (4 g) by melting together with glycidyl-PEG.

Absorption spectra of the dyes (S1-S3) prepared in examples 1-3 areshown in FIG. 8.

In order to covalently label target molecules the carbonyl moiety has tobe converted into an activated ester (reactive group R_(x)). Due tostate of the art, this can be carried out with N-hydroxysuccinimide inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) inaqueous solution or with dicyclohexylcarbodiimide (DCC) in organicsolution (FIG. 2, 1 f)

Example 10 Coupling of the NHS Activated pentamethine-spiro-cyanine(Compound S2) to Protein

0.2 mg activated dye are added while shaking to 1 mg of bovine serumalbumin in 1 ml 10 mM phosphate buffer, pH 8.0. After a reaction time of60 min, the coupling is stopped by the addition of 40 μl glycinesolution (10% m/v, in PBS-buffer, pH 7.4) and the excess of dye isremoved through dialysis or gelfiltration.

FIG. 9 shows the emission spectrum of the dye-conjugate obtained withdye S2 (example 2) as described in example 10.

In comparison, FIG. 10 shows the corresponding spectrum of a commonconjugate created under identical conditions. A band in the absorptionspectrum at about λ=600 nm due to dimer formation which leads to reducedfluorescence can clearly be seen.

The mobility of the fluorescent dye is reduced due to the influence ofthe bulky spiro-substituent, which leads to an increased photostability.FIG. 11 displays the photostability of the compound S3 preparedaccording to example 3, determined in 10 mM phosphate buffer, pH 7.4,compared to a commercial state-of-the-art heptamethine dye. Irradiationwas performed using a krypton-lamp and 20 mW/cm² at λ>400 nm.

By determination of the fluorescence of the S3-conjugate, according toexample 10, in comparison to a conjugate, created using a commoncyanine-dye, the results displayed in FIG. 12 are obtained. Theresulting fluorescence of the conjugates, created under identicalconditions, was determined at different incubation times afterseparation of the excess of the dye. The longer the incubation time, thehigher the label degree fluorophor:protein. The formation of a narrowmaximum is observed with the conjugate according to the state of theart, the resulting fluorescence decreases more than 50% at higherlabeling degrees due to self-quenching phemonena. In contrast,conjugates with the fluorescent dye according to the invention (examples2 and 10) show a substantially reduced tendency towards aggregation andthus exhibit higher fluorescence. The fluorescence of the conjugate withthe invention based fluorophor (example 10) after an incubation time of15 minutes is more than double as high as the one of the conjugate witha state-of-the-art fluorophor.

The spiro-substituent also causes a shielding of the central polymethinechain against the influence of the surroundings of the fluorophor. Asknown, many fluorophores exhibit a shift of their absorption-maximumtowards longer wavelengths and a simultaneous increase of fluorescencewith an increase of protein concentration. This is caused by the changedenvironment created by the protein and the reduced mobility of thefluorescent dye. This effect is minimized with the fluorescent dyeaccording to the invention. As shown in FIG. 13 (normalized display),there only is a minimal increase of fluorescence in the presence of highconcentrations of bovine serum albumin (BSA). The fluorescence of thecommercial fluorophor, in contrast, is highly dependent on theenvironment. Latter effect is a very strong disadvantage when analyzingsubstances in blood serum.

In table 2 the characteristic data of selected fluorescent dyesaccording to the invention are shown.

TABLE 1 type of chemical electrophilic group nucleophilic group bondageformed activated ester¹⁾ amine/aniline carboxamide acylazideamine/aniline carboxamide acylhalide amine/aniline carboxamideacylhalogene alkohole/phenole ester acylnitrile alkohole/phenole esteracylnitrile amine/aniline carboxamide aldehyde amine/aniline iminealdehyde/ketone hydrazine hydrazone aldehyde/ketone hydroxylamine oximealkylhalogene amine/aniline alkylamine alkylhalogene carbonic acidsester alkylhalogene thiole thio ether alkylhalogene alcohol/phenol etheralkylsulfonate thiole thio ether alkylsulfonate carbonic acids esteralkylsulfonate alcohol/phenol ester anhydride alcohol/phenol esteranhydride amine/aniline carboxamide arylhalogene thiole thiophenolearylhalogene amine arylamine aziridine thiole thioester boronate glycoleboronatester carbonic acids amine/aniline carboxamide carbonic acidsalcohol ester carbonic acids hydrazine hydrazide carbodiimide carbonicacids N-acyl-urea diazoalkane carbonic acids ester epoxide thiole thioether haloacetamide thiole thio ether halotriazine amine/anilineaminotriazine halotriazine alcohol/phenol triazinether imidoesteramin/aniline amidine isocyanate amine/aniline urea isocyanatealcohol/phenol urethane isothiocyanate amine/aniline thiourea maleimidethiole thio ether phosphoramidite alcohol phosphite triestersilylhalogene alcohol silyl ether sulfonic acid ester amine/anilinealkylamine sulfonic acid ester carbonic acids ester sulfonic acid esterthiole thio ether sulfonic acid ester alcohol ether sulfonic acid esteramine/aniline sulfonamide sulfonylchloride alcohol/phenol sulfonic acidester ¹⁾activated ester with the general structure —CO-W, where in W isa suitable leaving group, e.g., nitro-, fluoro-, chloro-, cyano-.trifluoromethyl-. tosyl- etc.

TABLE 2 charactareistic data of selected fluorescent dyes according tothe invention absorption emission- maximum maximum Compound (nm) (nm)S1*  557 572 S2*  656 672 S3*  756 772 S4** 612 638 S5** 650 676 S6**603 626 S7** 702 728 S8** 662 684 S9** 660 682 *determined in phospatebuffer, pH 7.4 **determined in ethanol

1. A fluorescent dye having a formula (I)

wherein X is

O, or

and T is

n′ is an integer which is 0, 1, 2, or 3 and W⁻ is a counter ion, whereinat least one of the substituents R₁, R₁₀, R₆-R₉ and R₁₈ to R₁₉ is achemical reactive group (Rx) for covalent coupling to a target moleculewhich is selected from the group consisting of:

wherein n is 1-8; and wherein at least one of the substituents R₁, R₄,R₁₀, R₁₃, R₆ to R₉ and R₁₈ to R₁₉ is a hydrophilic group to promotewater solubility selected from the group consisting of sulfonic acidsand sulfonates, having a formula

with r equal to 0 to 8 and M⁺ is a metal cation; and the othersubstituents R₁ to R₁₉ are independently selected from the groupconsisting of H, alkyl (C₁-C₁₀), alkoxy (C₁-C₁₀), trifluoromethyl,nitro, and halogen.
 2. A fluorescent dye according to claim 1, in whichat least one of the substituents R₁, R₁₀, and R₈, R₉, is a chemicalreactive group (Rx).
 3. A fluorescent dye according to claim 1, in whichat least one of the substituents R₁, R₄, R₈, R₉, R₁₀, R₁₃, R₁₈, R₁₉ is ahydrophilic group.
 4. A fluorescent dye according to claim 1, in whichthe counter ion W⁻ is selected from the group consisting of halogenide,tosylate and perchlorate.
 5. A fluorescent dye according to claim 1,having a formula:

where M⁺ is a metal cation, X, T, Rx and n′ are defined according toclaim
 1. 6. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, X, Y, Rx and n′ are defined according toclaim
 1. 7. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, T, Rx and n′ are defined according toclaim
 1. 8. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, Y, Rx and n′ are defined according toclaim
 1. 9. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, Rx, Y, r and n′ are defined according toclaim
 1. 10. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation; X, T, n and n′ are defined according toclaim
 1. 11. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, X, T, n and n′ are defined according toclaim
 1. 12. A fluorescent dye according to claim 1, having a formula:

where M⁺ is a metal cation, X, Y, n and n′ are defined according toclaim
 1. 13. A fluorescent dye according to claim 1, having a formula:

where X, Y, n and n′ are defined according to claim 1.